The expression of hepatitis B surface antigen (HBsAg) in Escherichia coli (E. coli) poses considerable difficulties, especially because of its four transmembrane domains. These domains are essential for the correct assembly and operation of HBsAg, which inherently produces lipoprotein micelles needing certain membrane-spanning structures. While the structures have important functions in post-translational operations, studies have shown that HBsAg can be produced without those domains or by replacing those domains with other viral sequences. By removing its transmembrane domains, this study investigates the expression of recombinant hepatitis B surface antigen (HBsAg). We synthesised and cloned the modified gene into a suitable expression vector, pET-20b (+), and then transformed it into E. coli BL21 (DE3). The aim was to create soluble HBsAg for diagnostic use and to simplify the expression of proteins with transmembrane domains. The results showed successful expression of soluble HBsAg in E. coli BL21(DE3), paving the way for potential diagnostic applications. This study provides insights into the challenges and strategies involved in producing transmembrane protein variants for various research purposes